This invention relates generally to a modified mammalian factor VIII having amino acid substitutions which reduce its immunogenicity and/or antigenicity as compared to the proteins from which they were derived or other factor VIII preparations such as human factor VIII.
Blood clotting begins when platelets adhere to the cut wall of an injured blood vessel at a lesion site. Subsequently, in a cascade of enzymatically regulated reactions, soluble fibrinogen molecules are converted by the enzyme thrombin to insoluble strands of fibrin that hold the platelets together in a thrombus. At each step in the cascade, a protein precursor is converted to a protease that cleaves the next protein precursor in the series. Co-factors are required at most of the steps.
Factor VIII circulates as an inactive precursor in blood, bound tightly and non-covalently to von Willebrand factor. Factor VIII is proteolytically activated by thrombin or factor Xa, which dissociates it from von Willebrand factor and activates its procoagulant function in the cascade. In its active form, the protein factor VIIIa is a cofactor that increases the catalytic efficiency of factor IXa toward factor X activation by several orders of magnitude.
People with deficiencies in factor VIII or antibodies against factor VIII who are not treated with factor VIII suffer uncontrolled internal bleeding that may cause a range of serious symptoms, from inflammatory reactions in joints to early death. Severe hemophiliacs, who number about 10,000 in the United States, can be treated with infusion of human factor VIII, which will restore the blood""s normal clotting ability if administered with sufficient frequency and concentration. The classical definition of factor VIII is that substance present in normal blood plasma that corrects the clotting defect in plasma derived from individuals with hemophilia A.
The development of antibodies (xe2x80x9cinhibitorsxe2x80x9d or xe2x80x9cinhibitory antibodiesxe2x80x9d) that inhibit the activity of factor VIII is a serious complication in the management of patients with hemophilia. Autoantibodies develop in approximately 20% of patients with hemophilia A in response to therapeutic infusions of factor VIII. In previously untreated patients with hemophilia A who develop inhibitors, the inhibitors usually develops within one year of treatment. Additionally, autoantibodies that inactivate factor VIII occasionally develop in individuals with previously normal factor VIII levels. Inhibitory antibodies (inhibitors) to factor VIII (fVIII) either develop as alloantibodies in hemophilia A patients following fVIII infusions or as autoantibodies in nonhemophiliacs (Hoyer, L. W. and D. Scandella, 1994, xe2x80x9cFactor VIII inhibitors: structure and function in autoantibody and hemophilia A patients,xe2x80x9d Semin.Hematol. 31:1-5). Antibodies to epitopes in the A2, ap-A3, and C2 domains within the A1-A2-B-ap-A3-C1-C2 fVIII molecule are responsible for all anticoagulant activity in most inhibitor plasmas (Prescott, R. et al., 1997, xe2x80x9cThe inhibitory antibody response is more complex in hemophilia A patients than in most nonhemophiliacs with fVIII autoantibodies,xe2x80x9d Blood 89:3663-3671; Barrow, R. T. et al., 2000, xe2x80x9cReduction of the antigenicity of factor VIII toward complex inhibitory plasmas using multiply-substituted hybrid human/porcine factor VIII molecules,xe2x80x9d Blood 95:557-561). The 18-kDa C2 domain, defined as residues Ser2173-Tyr2332 in single chain human fVIII, contains a phospholipid membrane-binding site that is necessary for the normal procoagulant function of fVIII. Human C2-specific anti-fVIII antibodies inhibit this interaction (Arai, M. et al., 1989, xe2x80x9cMolecular basis offactor-VIII inhibition by human antibodiesxe2x80x94antibodies that bind to the factor-VIII light chain prevent the interaction of factor-VIII with phospholipid,xe2x80x9d J. Clin. Invest. 83:1978-1984). Consistent with this, phospholipid protects fVIII from inactivation by fVIII inhibitors (Arai et al., supra; Barrowcliffe, T. W. et al., 1983, xe2x80x9cBinding to phospholipid protects factor VIII from inactivation by human antibodies,xe2x80x9d J. Lab. Clin. Med. 101:34-43). The C2 domain also contains part of the von Willebrand factor (vWf) binding site (Saenko, E. L. et al., 1994, xe2x80x9cA role for the C2 domain of factor binding to von Willebrand factor. J. Biol. Chem. 269:11601-11605; Saenko, E. L. and Scandella, D., 1997, xe2x80x9cThe acidic region of the factor VIII light chain and the C2 domain together form the high affinity binding site for von Willebrand factor,xe2x80x9d J. Biol. Chem. 272:18007-18014). Some inhibitors may act by interfering with this interaction (Shima, M. et al., 1995, xe2x80x9cCommon inhibitory effects of human anti-C2 domain inhibitor alloantibodies on factor VIII binding to von Willebrand factor,xe2x80x9d Br. J. Haematol. 91:714-721; Saenko, E. L. et al., 1996, xe2x80x9cSlowed release of thrombin-cleaved factor VIII from von Willebrand factor by a monoclonal and human antibody is a novel mechanism for factor VIII inhibition,xe2x80x9d J. Biol. Chem. 271:27424-27431; Gilles, J G. et al., 1999, xe2x80x9cSome factor VIII (FVIII) inhibitors recognize a FVIII epitope(s) that is present only on FVIII-vWf complexes,xe2x80x9d Thromb. Haemost. 82:40-45).
Patients can be managed by increasing the dose of factor VIII provided the inhibitor titer is low enough. However, often the inhibitor titer is so high that it cannot be overwhelmed by factor VIII. An alternative strategy is to bypass the need for factor VIII during normal hemostasis using factor IX complex preparations (for example, KONYNE(copyright), Proplex(copyright)) or recombinant human factor VIIIa. Additionally, since porcine factor VIII usually has substantially less reactivity with inhibitors than human factor VIII, a partially purified porcine factor VIII preparation (HYATE:C(copyright)) is used. Many patients who have developed inhibitory antibodies to human factor VIII have been successfully treated with porcine factor VIII and have tolerated such treatment for long periods of time. However, administration of porcine factor VIII is not a complete solution because inhibitors may develop to porcine factor VIII after one or more infusions.
Several preparations of human plasma-derived factor VIII of varying degrees of purity are available commercially for the treatment of hemophilia A. These include a partially-purified factor VIII derived from the pooled blood of many donors that is heat- and detergent-treated for viruses but contain a significant level of antigenic proteins; a monoclonal antibody-purified factor VIII that has lower levels of antigenic impurities and viral contamination; and recombinant human factor VIII, clinical trials for which are underway. Unfortunately, human factor VIII is unstable at physiologic concentrations and pH, is present in blood at an extremely low concentration (0.2 xcexcg/ml plasma), and has low specific clotting activity.
Hemophiliacs require daily replacement offactor VIII to prevent bleeding and the resulting deforming hemophilic arthropathy. However, supplies have been inadequate and problems in therapeutic use occur due to difficulty in isolation and purification, immunogenicity, and the necessity of removing the AIDS and hepatitis infectivity risk. The use of recombinant human factor VIII or partially-purified porcine factor VIII will not resolve all the problems.
The problems associated with the commonly used, commercially available, plasma-derived factor VIII have stimulated significant interest in the development of a better factor VIII product. There is a need for a more potent factor VIII molecule so that more units of clotting activity can be delivered per molecule; a factor VIII molecule that is stable at a selected pH and physiologic concentration; a factor VIII molecule that is less apt to cause production of inhibitory antibodies; and a factor VIII molecule that evades immune detection in patients who have already acquired antibodies to human factor VIII.
U.S. Pat. No. 6,180,371 to Lollar describes amino acid substitutions in the A2 domain of human factor VIII which alter the antigenicity of the resulting factor VIII molecules. The ""371 patent does not disclose or suggest specific amino acid substitutions in the C2 domain which reduces antigenicity or immunogenicity as compared to wild-type factor VIII or the corresponding recombinant factor VIII.
U.S. Pat. No. 5,859,204 to Lollar discloses the site specific replacement of amino acids in the 484-509 region of human factor VIII. More specifically, the ""204 patent teaches modified factor VIII with amino acid substitutions at positions 485, 487, 488, 489, 492, 495, 501 or 508 relative to the human protein. The ""204 patent does not disclose or suggest specific amino acid substitutions in the C2 domain which reduce antigenicity or immunogenicity as compared to wild-type factor VIII or the corresponding recombinant factor VIII.
U.S. Pat. No. 5,888,974 to Lollar et al. discloses hybrid procoagulant factor VIII produced by the isolation and recombination of human and other non-human factor VIII subunits or domains.
U.S. Pat. No. 5,744,446 to Lollar et al. describes hybrid factor VIII having amino acid substitutions in the A2 domain.
U.S. Pat. No. 5,663,060 to Lollar et al. describes hybrid factor VIII comprised of combinations of non-human and human heavy and light chain subunits. U.S. Pat. No. 5,583,209 describes nucleic acids encoding the hybrid factor VIII molecules in the ""060 patent.
U.S. Pat. No. 5,364,771 describes purified hybrid factor VIII comprised of human and porcine combinations of the heavy and light subunits. Also disclosed is human factor VIII with porcine A2 domain swapped for the human A2 domain.
U.S. Pat. Nos. 6,180,371; 5,888,974; 5,859,204; 5,744,446; 5,663,060; 5,583,209; and 5,364,771 (all of which are incorporated herein by reference) do not disclose substitutions or suggest specific amino acid substitutions in the C2 domain of factor VIII which reduce antigenicity or immunogenicity as compared to wild-type factor VIII or the corresponding recombinant factor VIII. Despite years of intensive research from laboratories around the world, it appears that the invention regarding the C2 domain of factor VIII described in detail herein has not been described or suggested elsewhere.
Pratt et al. (1999, xe2x80x9cStructure of the C2 domain of human factor VIII at 1.5 xc3x85 resolution,xe2x80x9d Nature 402:439-442) have reported the crystal structure of the C2 domain of human factor VIII at 1.5 xc3x85 resolution. Pratt et al. reported that the structure partly explains why mutations in the C2 region of factor VIII lead to bleeding disorders. In fact, 21 residues in the C2 region were reported to be sites of deleterious point mutations in patients with hemophilia A. For example, V2223 is known to be a position where a point mutation occurs and is associated with bleeding disorders. Thus, one of ordinary skill in the art would not expect V2223 to be a reasonable candidate for substitution to provide modified factor VIII for therapeutic activity. Indeed, Shima et al. report C2 binding antibody inhibitors interfere with factor VIII with respect to phospholipid and Von Willebrand factor binding. Thus, it is taught by Pratt et al. that C2 inhibitors, i.e., those related to some bleeding disorders in individuals with hemophilia A, interfere with the binding of the C2 domain to phospholipid and Von Willebrand factor. This conclusion, combined with their determination that M2199, F2200, L2251, L2252, V2223, and R2220 appear at the protein-phospholipid interface, suggests that mutation of these residues would lead to altered phospholipid and/or Von Willebrand binding along with an associated increase in bleeding disorders. It is not clear from these studies which amino acid residues and corresponding substitutions would lead to improved factor VIII molecules.
Unexpectedly it was discovered by the inventor of the instant invention that mutations in the 3 hydrophobic feet identified in the recently available x-ray structure for the C2 domain of factor VIII have reduced binding to inhibitory antibodies, improved properties, and/or reduced immunogenicity.
It is therefore an object of the present invention to provide a factor VIII that corrects hemophilia in a patient deficient in factor VIII or having inhibitors to factor VIII.
It is a further object of the present invention to provide methods for treatment of hemophiliacs.
It is still another object of the present invention to provide a factor VIII that is stable at a selected pH and physiologic concentration.
It is yet another object of the present invention to provide a factor VIII that has greater coagulant activity than human factor VIII.
The present invention generally relates to compositions comprising recombinant mammalian factor VIII. The composition of the invention comprise isolated, purified recombinant mammalian factor VIII molecules with coagulant activity wherein the recombinant factor VIII has amino acid substitutions in the C2 domain which reduce antigenicity as compared to the proteins from which they were derived or other factor VIII preparations. DNA sequences encoding the novel compositions of the invention as well as methods of producing the novel compositions comprising factor VIII are also provided. Methods of treating patients in need of treatment with factor VIII are also within the scope of this invention.
A first embodiment of the invention provides novel compositions comprising recombinant mammalian factor VIII with amino acid substitution(s) in the C2 domain. The amino acid substitution(s) in the C2 domain of the modified recombinant factor VIII reduce the anticoagulant activity of inhibitory antibodies as compared to the proteins from which they were derived or other available factor VIII preparations. The novel composition of this embodiment have coagulant activity and reduced binding to inhibitory antibodies. Substitutions at residues that participate in binding of fVIII to phospholipid membranes and/or to inhibitory antibodies are preferred. Preferred substitutions at positions homologous to human factor VIII include, but are not limited to, Met2199, Phe2200, Val2223, Leu2251, and Leu2252. The novel compositions of this embodiment can be a single mutant, a double mutant, a triple mutant, or a quadruple mutant. Examples of amino acid substitutions of the invention include, but are not limited to, Met2199Ile, Phe2200Leu, Leu2252Phe, Met2199Ile/Phe2200Leu,Val2223Ala/Lys2227Glu, Met2199Ile/Phe2200Leu/Va2223 Ala/Lys2227Glu, all of which are referenced to the human factor VIII numbering system wherein amino acid number 1 is the amino terminal alanine of mature factor VIII. Substitutions in either recombinant porcine or human factor VIII are preferred.
A second embodiment of the invention provides novel hybrid factor VIII compositions comprising recombinant factor VIII with amino acid substitution(s) in the C2 domain. The novel compositions of this embodiment are constructed by preparing hybrid factor VIII with amino acid substitutions in the C2 domain. The other domains of factor VIII may be derived from a variety of mammals such as human, mouse, pig, rat, and canine and so on. The novel compositions of this embodiment have coagulant activity and reduced binding to inhibitory antibodies. Examples of amino acid positions that can be mutated to provided the novel compositions of this embodiment include, but are not limited to, Met2199, Phe2200, Val2223, Leu2251, and Leu2252, all of which are referenced to human factor VIII. Examples of amino acid substitutions in the C2 domain encompassed within this embodiment include, but are not limited to, Met2199Ile, Phe2220Leu, Leu2252Phe, Met2199Ile/Phe2200Leu,Val2223Ala/Lys2227Glu, Met2199Ile/Phe2200Leu/-Val2223Ala/Lys2227Glu, all of which are referenced to the human factor VIII.
Another embodiment of the invention provides DNA sequences comprising coding sequences for the novel compositions of the invention. Yet another embodiment of the invention provides methods of producing the novel compositions of the invention.
The invention also provides a method for reducing the immunogenicity of factor VIII molecules as well as recombinant factor VIII with reduced immunogenicity produced by the method. In particular, modified recombinant factor VIII molecule and methods of making such molecules with reduced immunogenicity that have substitutions in the C2 domain are described.
Also provided are pharmaceutical compositions and methods for treating patients having factor VIII deficiency comprising administering recombinant factor VIII and hybrid version thereof.